Adeno-associated virus (AAV) is a non-pathogenic, replication defective virus. Recombinant AAV (rAAV) shows promise as a viral vector for gene therapy. rAAV has a simple structure consisting of 3 capsid proteins and a single-stranded DNA genome, and does not express virally encoded proteins. Despite its nonpathogenic and replication defective nature, there are intracellular virus-host interactions mediated by viral genome DNA (i.e., single-stranded DNA with hairpins). Although there has been substantial progress in the application of this vector system to treat various diseases, the role of intracellular virus-host interactions in rAAV vector biology and host cellular biology in vivo (in animals and humans) remains largely unknown. Our ultimate goal in this proposal is to substantially understand complex rAAV vector biology in vivo and cellular biology of somatic cells in transduced tissues. Toward achieving this goal, we will specifically focus on interactions between rAAV genomes, host chromosomal DNA and host cellular DNA repair machinery. We recently discovered that, if rAAV vector genome inverted terminal repeat (ITR) hairpin structures are not resolved by a specific host cellular endonuclease activity(ies), an unusual form, no-end double-stranded linear monomer rAAV vector genomes, accumulate in transduced cells in mice. Based on this observation, we hypothesize that activation of this cellular endonuclease activity via virus-host cell interactions plays a central role in rAAV vector genome processing and establishment of stable transduction in vivo. In the proposal, we will take advantage of the formation of this unusual no-end vector genome structure to identify cellular factors involved in vector-host cell interactions leading to AAV-ITR hairpin loop opening (aim 1). Once we identify cellular factors, we will investigate how the cellular factors and vector genomes interact in a network and how this network is activated by rAAV vectors (aim 2). In addition, we will investigate how rAAV vector genomes, host chromosomal DNA and the cellular factors interact, resulting in vector genome integration in various tissues in vivo (aim 3). Finally, because such cellular factors constitute DNA repair machinery and because DNA repair efficacy decreases and genomic instability increases with age, we will investigate how age affects vector and host cellular biology in vivo (aim 4). The proposed project should substantially contribute to building an intellectual foundation for successful human gene therapy.